The present invention relates to a method of controlling sprout formation in plants and parts thereof including vegetative storage organs.
Potato tubers are of major economic importance. They represent a carbohydrate resource for many diets and are used as a basis for a variety of processed products. Besides starch, tubers contain high-quality proteins, substantial amounts of vitamins, minerals and trace elements. Continuous production of potato tubers throughout the year is impossible in most regions where potatoes are grown. As a consequence storage of the harvested tubers is required.
One of the potentially most damaging phenomena during storage is premature sprouting. Long term storage involves cooling, forced ventilation and use of chemical sprouting suppressants. The problems directly linked to long term storage are manifold.
Cooling, usually done in Northern Europe by ventilation with air at ambient temperature is one of the methods to inhibit sprouting. Apart from the associated costs, longer term cooling at 4xc2x0 C. gives rise to the problems of cold sweetening and melanisation (darkening).
Chemical sprouting suppressants are currently the only possibility for inhibiting sprouting in potatoes destined for processing and fresh consumption, since low temperature storage leads to unacceptable accumulation of reducing sugars. However, in recent years, questions have arisen as to the environmental and nutritional impact of chemical suppressants such as chlorinated hydrocarbons. There is therefore a real need for an alternative method of controlling sprouting in vegetative storage organs such as tubers.
An alternative approach to delay sprouting would be the use of transgenic plants with a prolonged quiescence period. Sprouting of potato tubers involves several independent steps which might be targets for genetic engineering. The first step is the mobilisation of reserves, mainly starch. Starch breakdown occurs in amyloplasts and is mediated by starch phosphorylase and/or amylases. In the next step following starch breakdown, the resultant hexoses and/or hexose-phosphates have to be exported from amyloplasts. After transfer into the cytosol the produced hexoses and hexose-phosphates are distributed between glycolysis and sucrose synthesis. The third step is the formation of sucrose in the cytosol. Sucrose synthesis is energy dependent thus glycolysis and respiration are required. The fourth step is the transport of sucrose to the developing sprout. Finally the imported sucrose is utilised in the sprout to support growth and development.
We have now developed a means of controlling sprouting in vegetative storage organs such that sprouting may be turned off and on without any undesirable side effects such as yield loss. This new method involves the targeted expression of genes resulting in the disruption of sprouting in combination with gene switch technology to restore sprouting when required.
According to a first aspect of the present invention there is provided a method for the selective induction or suppression of sprouting in a plant comprising incorporating, preferably stably incorporating, into the genome of said plant by transformation a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and optionally to a transcription terminator region whereby the DNA sequence(s) in said first polynucleotide sequence is expressed during dormancy of the vegetative organ derived from said transgenic plant resulting in effective suppression of sprouting and the said suppression is neutralised by inducing expression of the DNA sequence(s) in said second polynucleotide sequence from said controllable promoter region by external application of an inducing substance such that restoration of sprouting of said vegetative storage organ is dependent on the application of the inducer.
As used herein the term xe2x80x9ctissue or organ selective promoter regionxe2x80x9d denotes those promoter regions which yield preferential expression of the DNA sequence(s) of interest in the desired tissue or organs.
The DNA sequences in the DNA construct may be endogenous or heterologous with respect to the transformed host.
Examples of DNA sequences which may be used in the method of the present invention to control sprouting include those DNA sequences coding for proteins involved in the mobilisation of reserves during dormancy such as the breakdown of storage compounds e.g starch breakdown, i.e starch phosphorylase, amylase (e.g. xcex1 or xcex2 amylase) and maltase; e.g in glycolysis and subsequent metabolism e.g phosphofructokinase, hexokinase; in sucrose biosynthesis e.g sucrose synthase; in the transport of reserves during dormancy such as in phloem loading e.g ATPase; in long distance phloem transport and in phloem unloading e.g inorganic pyrophosphorylase (iPPase); and in the utilisation of reserves during dormancy such as in assimilate breakdown e.g the breakdown of sucrose in the growing sprout, i.e invertase; and in the utilisation of assimilates e.g utilisation of sucrose-derived metabolites, in the provision of energy required for sprout formation e.g. DNA sequences coding for proteins involved in mitochondrial function such as in respiration, such as mitochondrial enzymes and transport proteins such as translocators e.g. adenine nucleotide translocator (ANT) and malate oxoglutarate translocator (MOT) and inhibitors thereof such as uncoupling proteins. Examples of useful DNA sequences also include any other sequences which are involved in potato sprouting
Examples of preferred DNA sequences which may be used in the method of the present invention to control sprouting include those resulting in the production of sense, anti-sense or partial sense sequence(s) to, and/or coding for, proteins involved in the mobilisation and/or utilisation of sucrose, in potato sprouting and in mitochondrial function such as in respiration.
Examples of particularly preferred DNA sequences include those coding for an invertase derived from plant, bacterial or fungal sources e.g. from yeast, a pyrophosphatase derived from plant, bacterial or fungal sources and proteins involved in mitochondrial function such as MOT and ANT derived from plant, bacterial or fungal sources which are described hereinafter.
Suppression of sprouting may be achieved in a variety of ways. The first DNA sequence(s) may be expressed during dormancy of the vegetative storage organ and then down-regulated when sprouting is desired. When sprouting is desired expression of the second DNA sequence(s) is turned on leading to down regulation of the first DNA sequence and consequently restoration of sprouting.
Down regulation of a desired DNA sequence(s) may be achieved using methods well known in the art such as, for example, by use of repressor proteins, sense, anti-sense, partial-sense, and expression of a complementary protein. Examples of suitable operator/repressor systems include for example the lac, tet or lambda 434 systems and mutants thereof such as the Lac Ixcex94 His mutant (Lehming, N., Sartoris, J., Niemoeller, M., Genenger, G., v. Wilcken-Bergman, B. and Muller-Hill, Benno (1987), EMBO J. 6(10) 3145-3153xe2x80x94where the mutant has a change in amino acid 17 of Lac I altering tyrosine for histidine). Alternatively, an Amplicon(trademark) may be used to down-regulate genes (Angell, S. M., Baulcombe, D. C., (1997) 16, 3675-3684). In this regard, the cDNA of replicating potato virus (PVX) RNA which has a transgene inserted therein is used whereby transiently expressed RNA sharing homology with the transgene is suppressed.
Alternatively, expression of the DNA sequence(s) in the first polynucleotide sequence may result in the production of a sense, anti-sense or partial-sense sequence(s) which acts to suppress a gene involved in sprouting or in the expression of an Amplicon(trademark). In this case sprouting is restored by switching on expression of the DNA sequence(s) in the second polynucleotide sequence which results in production of the protein or a corresponding protein to that, the production of which was suppressed by the sense, anti-sense or partial-sense sequence(s) in the first DNA sequence. Sprouting may also be restored by means of a suitable operator/repressor system.
Where either or both of the polynucleotide sequences in the construct comprise more than one DNA sequence it is preferable that they are not identical to avoid any co-suppression effects.
Expression of the DNA sequence(s) in the first polynucleotide sequence is under the control of a tissue or organ selective promoter to ensure targeted expression of the DNA sequence whereby expression is induced in an organ or tissue specific manner. Examples of tissue selective promoters include phloem selective promoters e.g. the rolC promoter, and examples of organ selective promoters include tuber specific promoters, such as the patatin promoter. The use of tissue or organ selective promoters such as the rolC and tuber promoters is particularly preferred.
The DNA sequence(s) in the second polynucleotide sequence of the construct is under the control of a controllable promoter region.
As used herein the term xe2x80x9ccontrollable promoter regionxe2x80x9d includes promoters which may be induced chemically. The use of a promoter sequence which is controlled by the application of an external chemical stimulus is most especially preferred. The external chemical stimulus is preferably an agriculturally acceptable chemical, the use of which is compatible with agricultural practice and is not detrimental to plants or mammals.
The controllable promoter region most preferably comprises an inducible switch promoter system as such as, for example, a two component system such as the alcA/alcR gene switch promoter system described in our published International Patent Application No. WO 93/21334; the GST promoter as described in our published International Patent Application Nos. WO 90/08826 and WO 93/031294; and the ecdysone switch system as described in our published International Patent Application No. WO 96/37609, the teachings of which are incorporated herein by reference. Such promoter systems are herein referred to xe2x80x9cswitch promotersxe2x80x9d. The switch chemicals used in conjunction with the switch promoters are agriculturally acceptable chemicals making this system particularly useful in the method of the present invention. In the case of the alcA/alcR promoter switch system the preferred chemical inducer is ethanol in either liquid or more preferably in the vapour form. One of the main advantages of the use of ethanol vapour is that only small quantities of ethanol are required and that high levels of expression are achieved. Full details of switch chemicals are provided in the patent applications listed immediately above.
Suitable transcription terminators which may be used are also well known in the art and include for example the nopaline synthase terminator and octopine synthase terminators. The promoter is most desirably a late tuber specific promoter which is active late in the dormancy period i.e just before sprouting.
The controllable promoter region for use in the method of the present invention is preferably the GST or alcA/alcR promoter switch system. Restoration of sprouting is preferably achieved using switchable antisense or switchable sense or partial sense methods as is described more fully herein or alternatively by use of an Amplicon(trademark) or by means of a suitable operator/repressor system. Down-regulation of gene activity due to partial sense co-suppression is described in our International Patent Application No. WO 91/08299 the teachings of which are incorporated herein and this may be avoided if necessary by using gene sequences derived from different organisms.
According to a second aspect of the present invention there is provided a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and a transcription terminator region wherein said first polynucleotide sequence comprises a DNA sequence coding for a protein involved in mobilisation and/or utilisation of sucrose and said second polynucleotide sequence comprises a DNA sequence which is a sense, an anti-sense or partial sense sequence corresponding to said protein or a DNA sequence which is capable of causing suppression of said protein.
According to a third aspect of the present invention there is provided a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and a transcription terminator region wherein said first polynucleotide sequence comprises a first DNA sequence coding for a protein involved in mobilisation and/or utilisation of sucrose and a further DNA sequence coding for an operator sequence operably linked to the first DNA sequence and the second polynucleotide sequence comprises a DNA sequence coding for a repressor protein capable of binding to said operator sequence.
According to a fourth aspect of the present invention there is provided a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and a transcription terminator region wherein said first polynucleotide comprises a DNA sequence(s) which is a sense, anti-sense or partial sense sequence corresponding to a protein involved in potato sprouting or a DNA sequence which is capable of causing suppression of a protein involved in potato sprouting and said second polynucleotide sequence comprises a DNA sequence(s) coding for a protein involved in potato sprouting.
According to a fifth aspect of the present invention there is provided a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and a transcription terminator region wherein said first polynucleotide comprises a first DNA sequence(s) which is a sense, anti-sense or partial sense sequence corresponding to a protein involved in potato sprouting or a DNA sequence which is capable of causing suppression of a protein involved in potato sprouting and a further DNA sequence coding for an operator sequence operably linked to the first DNA sequence and said second polynucleotide sequence comprises a DNA sequence(s) coding for a repressor protein capable of binding to said operator sequence.
According to a sixth aspect of the present invention there is provided a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and a transcription terminator region wherein said first polynucleotide comprises a DNA sequence(s) which is a sense, anti-sense or partial sense sequence corresponding to a protein involved in mitochondrial function or a DNA sequence which is capable of causing suppression of a protein involved in mitochondrial function and said second polynucleotide sequence comprises a DNA sequence(s) coding for a protein involved in mitochondrial function.
According to a seventh aspect of the present invention there is provided a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and a transcription terminator region wherein said first polynucleotide comprises a first DNA sequence(s) which is a sense, anti-sense or partial sense sequence corresponding to a protein involved in mitochondrial function or a DNA sequence which is capable of causing suppression of a protein involved in mitochondrial function and a further DNA sequence coding for an operator sequence operably linked to the first DNA sequence and said second polynucleotide sequence comprises a DNA sequence(s) coding for a repressor protein capable of binding to said operator sequence.
We have found the following combination of DNA sequences to be particularly suitable for use in the method of the invention: by placing a DNA sequence coding for an invertase under the control of a phloem selective promoter such as the rolC promoter, it is possible to target expression of the DNA sequence to the phloem and effectively repress sprouting and to then restore sprouting by switching on a DNA sequence coding for invertase anti-sense using the alcA/alcR chemical switch promoter. Sucrose concentration in the phloem from the leaf is so high that the effects of invertase expression are effectively swamped avoiding any unwanted side effects. This contrasts with the situation in the sprout phloem where expression of invertase has a dominant effect with the result that sucrose is broken down and sprouting is effectively inhibited.
A further useful combination is a DNA sequence coding for an inorganic pyrophosphatase (iPPase) under the control of a tuber promoter. Uptake of sucrose and transport in the phloem is an energy requiring process and by inhibiting the provision of energy by expressing the DNA sequence coding for inorganic pyrophosphatase it is possible to inhibit the uptake process. The inhibition can be reversed by using, for example, an alcA/alcR chemically induced switch promoter to switch on a DNA sequence coding for an antisense, sense or partial sense sequence to iPPase and sprouting is restored. Again the use of a tissue or organ selective promoter ensures that the inhibition of sucrose uptake and transport in the phloem does not occur in the whole plant but only in the tuber thereby minimising any deleterious effects in the plant.
In both cases, an alternative means of restoring sprouting is by the use of an Amplicon(trademark) where transiently expressed RNA sharing homology with the transgene is suppressed. Such a transgene could, for example, be a cDNA for an invertase or iPPase. A further alternative means of restoring sprouting is by the use of a suitable operator/repressor system.
We have also found that by selectively inhibiting the provision of energy required for sprout growth and development in the tuber by placing a DNA sequence coding for sense, antisense or partial sense to a DNA sequence coding for a protein involved in mitochondrial function, such as the adenosine nucleotide translocator protein (ANT) or mitochondrial oxoglutarate translocator (MOT), under the control of a tuber selective promoter sprouting may be inhibited without unwanted side effects. Alternatively, a DNA sequence which causes suppression of such proteins may be used. One way in which reversal of the inhibition may be achieved is by switching on expression of a second DNA sequence the product of which is complementary to the first DNA sequence, for example a DNA sequence coding for ANT derived from a different source preferably from Arabidopsis may be used to counteract the effect of the ANT antisense expression. In the case of MOT a suitable complementary sequence may be derived from Panicum miliaceum as is described by Taniguchi and Sugiyama in Plant Molec. Biol. 30, 51-64 (1996). Alternatively, a suitable operator/repressor system may be used to reverse inhibition. As above the alcA/alcR chemical switch promoter may be used. The above examples are described more fully herein.
According to some embodiments of the present invention the first polynucleotide sequence comprises a further DNA sequence coding for an operator sequence operably linked to the first DNA sequence and the second polynucleotide sequence comprises a DNA sequence coding for a repressor capable of binding to the operator sequence under the control of a switch promoter such that application of the inducer results in expression of the DNA sequence coding for the repressor which subsequently binds to the operator and expression of the first DNA sequence in the first polynucleotide sequence is switched off. An example of such a system is the lactose operator and repressor protein as is described in published International patent Application No. WO 90/08830. Other examples include the tetracycline and lambda 434 operator/repressor systems.
Plant cells may be transformed with recombinant DNA constructs according to a variety of known methods for example, Agrobacterium Ti plasmids, electroporation, microinjection and by microprojectile gun. The transformed cells may then, in suitable cases, be regenerated into whole plants in which the new nuclear material is incorporated, preferably stably incorporated, into the genome. Both transformed monocotyledonous and dicotyledenous plants may be obtained in this way.
According to an eighth aspect of the present invention, there is provided a plant cell transformed with any one of the DNA constructs defined above.
According to a nineth aspect of the present invention there is also provided a whole plant transformed with a DNA construct according to the above aspects of the present invention wherein said DNA construct is incorporated, preferably stably incorporated, into the genome of said plant.
The invention still further includes, according to a tenth aspect of the present invention, progeny of the plants of the preceding paragraph which progeny comprise a DNA construct according to the above aspects of the present invention incorporated, preferably stably incorporated, into their genome and the seeds and tubers of such plants and such progeny.
The method of the present invention is particularly suitable for controlling sprouting in potato tubers.
In a preferred embodiment the invention provides a method for the selective induction or suppression of sprouting in potatoes comprising stably incorporating into the genome of said potato by transformation a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and optionally to a transcription terminator region whereby the DNA sequence(s) in said first polynucleotide sequence is expressed during dormancy of the tuber derived from said transgenic potato resulting in effective suppression of sprouting and the said suppression is neutralised by inducing expression of the DNA sequence(s) in said second polynucleotide sequence from said controllable promoter region by external application of an inducing substance such that restoration of sprouting of said tuber is dependent on the application of the inducer.
We have also identified five particularly preferred DNA sequences which we believe may also be especially useful in the method of the present invention. We have identified these DNA sequences as being induced during tuber storage and we have designated these as 16-3 (sequence 2), 16-8 (sequence 3), 10-1 (sequence 4) and AC4 (sequence 5), M-1-1 (MOT) (sequence in FIG. 19) and a MOT variant (sequence 6xe2x80x94having an EMBL Accession number X99853). The DNA sequences and their isolation are described fully in the accompanying examples. The present invention therefore provides, according to a further aspect, the use of all or part of the DNA sequences from clones 16-3, 10-1, AC4, 16-8, M-1-1 and the MOT variant in a method according to the invention to control sprouting in plants.
The DNA sequences of 16-3, 16-8, AC4 and M-1-1 are believed to be new and a twelfth aspect of the present invention extends to polynucleotides comprising nucleotides 1 to 870 in sequence 2 (corresponding to 16-3), nucleotides 1 to 712 in sequence 3 (corresponding to 16-8) or nucleotides 1 to 386 in sequence 5 (corresponding to AC4) or nucleotides 1 to 1351 in sequence FIG. 19 (corresponding to M-1-1 encoding a MOT) and further to the protein products encoded thereby and to those proteins having a substantially similar activity and having an amino acid sequence which is at least 85% similar to the said product. It is preferred that the degree of similarity is at least 90% and it is more preferred that the degree of similarity is 95% and it is most preferred that the degree of similarity is 97%.
A particularly preferred embodiment of the polynucleotides consists of nucleotides 55 to 751 in sequence 2, nucleotides 87 to 473 in sequence 3, and to nucleotides 192 to 164 in FIG. 19 and further to the translation products encoded thereby and to those proteins having a substantially similar activity and having an amino acid sequence which is at least 85% similar to the said product. It is preferred that the degree of similarity is at least 90% and it is more preferred that the degree of similarity is 95% and it is most preferred that the degree of similarity is 97%.
As used herein the term xe2x80x9cdegree of similarityxe2x80x9d is used to denote sequences which when aligned have similar (identical or conservatively replaced) amino acids in like positions or regions, where identical or conservatively replaced amino acids are those which do not alter the activity or function of the protein as compared to the starting protein. For example, two amino acid sequences with at least 85% similarity to each other have at least 85% similar (identical or conservatively replaced amino residues) in a like position when aligned optimally allowing for up to 3 gaps, with the proviso that in respect of the gaps a total of not more than 15 amino acid resides is affected. The degree of similarity may be determined using methods well known in the art (see, for example, Wilbur, W. J. and Lipman, D. J. xe2x80x9cRapid Similarity Searches of Nucleic Acid and Protein Data Banks.xe2x80x9d Proceedings of the National Academy of Sciences USA 80, 726-730 (1983) and Myers E. and Miller W. xe2x80x9cOptimal Alignments in Linear Spacexe2x80x9d. Comput. Appl. Biosci. 4:11-17(1988)). One programme which may be used in determining the degree of similarity is the MegAlign Lipman-Pearson one pair method (using default parameters) which can be obtained from DNAstar Inc, 1228, Selfpark Street, Madison, Wis., 53715, USA as part of the Lasergene system.
According to a thirteenth aspect of the present invention there is provided polynucleotide sequence(s) encoding a protein having a substantially similar activity to that encoded by nucleotides provided in sequences 2, 3 and 5 and FIG. 19, which polynucleotide is complementary to one which still hybridises with the sequence comprised by that provided in sequences 2, 3 or 5 or FIG. 19 when incubated at or between low and high stringency conditions. In general terms, low stringency conditions can be defined as 3xc3x97SSC at about ambient temperature to about 65 C. and high stringency conditions as 0.1xc3x97SSC at about 65xc2x0 C. SSC refers to the buffer 0.15M NaCl, 0.015M trisodium citrate and 3xc3x97SSC is three times as strong as SSC and 0.1xc3x97SSC is one tenth of the strength of SSC.
The invention further provides polynucleotide sequence(s) encoding a protein having a substantially similar activity to that encoded by nucleotides 55 to 751 in sequence 2, nucleotides 87 to 473 in sequence 3, or to nucleotides 192 to 1164 in FIG. 19, which polynucleotide is complementary to one which still hybridises with the sequence comprised by nucleotides 55 to 751 in sequence 2, nucleotides 87 to 473 in sequence 3, or to nucleotides 192 to 1164 in FIG. 19 when incubated at or between low and high stringency conditions. In general terms, low stringency conditions can be defined as 3xc3x97SSC at about ambient temperature to about 65 C. and high stringency conditions as 0.1xc3x97SSC at about 65xc2x0 C. SSC refers to the buffer 0.15M Na Cl, 0.015M trisodium citrate and 3xc3x97SSC is three times as strong as SSC and 0.1xc3x97SSC is one tenth of the strength of SSC.
The polynucleotides according to the present invention depicted in sequences 2, 3 and 5 and FIG. 19 may be operably linked to a promoter region which may be homologous or heterologous to the polynucleotide and the present invention extends to such constructs. The present invention also extends to a DNA construct comprising said polynucleotides further comprising a region encoding a peptide which is capable of targeting the translation products of the polynucleotide to desired cellular or sub-cellular locations. The invention further provides a vector comprising said polynucleotide sequence as described in sequences 2, 3, 5 and FIG. 19 preferably operably linked to a promoter region and optionally to a transcription terminator and or a targeting sequence as described above.
The sequences provided herein for 16-3 (SEQ ID NO. 2), 16-8 (SEQ ID NO. 3), 10-1 (SEQ ID NO. 4), AC-4 (SEQ ID NO. 5), M1-1 (SEQ ID NO. 27) and the MOT variant are cDNA sequences and may, according to a further aspect of the present invention, be used as probes for the isolation and identification from genomic libraries of sequences upstream of the 5xe2x80x2 region which contain the natural promoter region. The promoter region may then be identified, isolated and sequenced.
According to a fifteenth aspect of the present invention there is provided a host cell transformed with a DNA construct comprising a polynucleotide sequence as described in sequences 2, 3 and 5 and FIG. 19 or a vector described above comprising said polynucleotide sequence. The host cell is preferably a plant cell as described previously and the present invention extends also to whole plants having incorporated, preferably stably incorporated, into their genome a polynucleotide sequence, DNA construct or vector as described above, and to seeds, tubers and progeny of said plants.
According to a sixteenth aspect of the present invention there is provided a DNA construct comprising a polynucleotide sequence comprising a switch promoter system operably linked to a polynucleotide sequence comprising a sense, antisense or partial sense transcription construct wherein when expression of said polynucleotide sequence is switched on from the switch promoter the resulting expression of said sense, antisense or partial sense sequence leads to down regulation of the expression of a further polynucleotide sequence encoding a transgene.
In a seventeenth aspect the present invention provides a method of controlling the expression of a transgene comprising transforming a host cell with a DNA construct comprising a switch promoter system operably linked to a polynucleotide sequence comprising a sense, antisense or partial sense transcription construct, and a further DNA construct comprising a coding sequence coding for a transgene and controlling expression of the polynucleotide sequence from said switch promoter such that the resulting expression of the said sense, antisense or partial sense construct leads to down regulation of the expression of said transgene.
As used herein the term xe2x80x9ctransgenexe2x80x9d is used to denote a gene which is foreign or heterologous to the transformed host cell.
In a preferred embodiment of the above aspects the present invention provides a DNA construct comprising the alcA/alcR switch promoter operably linked to a polynucleotide sequence comprising a sense, antisense or partial sense transcription construct.
The present invention also extends to a vector comprising said DNA constructs according to the above aspects of the invention.
According to an eighteenth aspect of the present invention there is provided a host cell transformed with a DNA construct comprising a polynucleotide sequence comprising a switch promoter which may be switched on by the application of a chemical stimulus operably linked to a polynucleotide sequence comprising a sense, antisense or partial sense transcription construct wherein when expression of said polynucleotide sequence is switched on from the switch promoter the resulting expression of said sense, antisense or partial sense sequence leads to down regulation of the expression of a further polynucleotide sequence encoding a transgene.
The host cell is preferably a plant cell as described previously and the present invention extends also to whole plants derived therefrom having incorporated, preferably stably incorporated, into their genome a polynucleotide sequence, DNA construct or vector as described above, and to seeds, tubers and progeny of said plants.
The use of switch promoter systems to control expression of the sense, antisense or partial sense construct has many applications. Down-regulation of a gene, the expression of which gives rise to a lethal or inhibitory effect may be controlled using switchable sense, antisense or partial sense to facilitate the identification of suitable herbicide targets. Switchable down regulation using sense, antisense or partial sense sequences may also be used to identify mechanisms of cell ablation.
The present invention therefore provides according to a nineteenth aspect a method of identifying a site which may be a suitable target for interaction with a herbicide comprising the steps of transforming a plant with a polynucleotide sequence comprising a first DNA sequence which is capable of affecting the expression of DNA at said target site wherein expression of said first DNA sequence is under the control of a switch promoter; controlling expression of said DNA sequence from said switch promoter such that the expression of the DNA coding for the herbicide target site is down regulated and determining the effects of said down regulation on the plant viability.
The types of effects which would be monitored include the time period for which down regulation at the target site must be maintained and what level of down regulation is required and on the basis of the results obtained it can be decided whether the target site would be suitable as a target site for a herbicide.
We have most unexpectedly found that the STLS-1 leaf promoter sequence acts as an enhancer of gene expression in tubers and the use of the STLS-1 sequence as an enhancer of gene expression in tubers forms a further aspect of the present invention.
In a twentieth aspect the present invention therefore provides a method of enhancing gene expression in tubers comprising transforming a tuber plant cell with a polynucleotide sequence comprising a DNA sequence coding for all or part of the STLS-1 leaf promoter operably linked to a further promoter region.
The STLS-1 leaf promoter is known in the art (Eckes et al (1986) Mol. Gen. Genet. 205, 14-22) and is described in the accompanying examples. All or part of the DNA sequence coding for the STLS-1 leaf promoter may be used as an enhancer according to the invention. Active fragments of STLS-1 may be identified using techniques well known in the art such as restriction enzyme digestion followed by analysis of enhancement of gene expression of the fragments thus obtained. The STLS-1 promoter sequence may be inserted either upstream i.e. at the 5xe2x80x2 end or downstream i.e. at the 3xe2x80x2 end of the further promoter region. Insertion of the STLS-1 sequence upstream of the promoter region is especially preferred. In a particularly preferred embodiment of this aspect of the invention the STLS-1 sequence is inserted upstream of the 35S CaMV promoter.
In a twenty-first aspect the present invention provides tubers, which are preferably potato tubers, derived from transgenic plants which do not sprout unless treated with a chemical inducer.
According to a twenty-second aspect of the present invention, there is provided a polynucleotide sequence comprising all or part of at least one of the sequences depicted in FIG. 25, 26 or 27 and polynucleotides having the same function as the sequence which is depicted in FIG. 25, 26 or 27 which polynucleotide is complementary to one which still hybridises with the sequence comprised by that provided in FIG. 25, 26 or 27 when incubated at or between low and high stringency conditions. Such sequences are preferably tuber specific promoters.
According to a twenty-third aspect of the present invention, there is provided a method of controlling gene expression of a plant or a part thereof comprising transforming a plant cell with a chemically inducible plant gene expression cassette comprising a first promoter operatively linked to a regulator sequence derived from the alc R gene and a controllable promoter derived from the alc A gene promoter operatively linked to a target gene, wherein the controllable promoter is activated by the regulator protein in the presence of alcohol vapour thereby causing expression of the target gene.